Nacently replicating strands of SV40 DNA have been isolated and cloned as an approach to the general problem of isolating mammalian origins of DNA replication. The analysis has demonstrated that rearrangement of the cloned DNA occurs with such high frequency that the general application of this method is not yet feasible. From the experiments, we have also found that DNA fork migration in bidirectionally replicating SV40 occurs at remarkably uniform rates. Of 33 clones analyzed, each SV40 fork had proceeded no more than 400 nucleotides further than the other and most were within 200 nucleotides of each other. In other experiments, a deletion of 18 base pairs in the 27 base pair palindromic sequence at the SV40 replication origin has been isolated. A DNA fragment including this deletion was hybridized to the corresponding wild-type DNA fragment generating heteroduplex molecules in which the palindromic sequence is forced into the stem and loop form. A series of experiments have been performed with the heteroduplex molecules to study the function of DNA in the stem-loop form.